Not applicable.
The prognosis of patients with malignant brain tumors is poor. Standard therapy, including surgery, radiation, and chemotherapy has proven ineffective in the majority of cases. One attempt to improve this grim clinical outlook has resulted from the discovery that many brain tumors over express the transferrin (xe2x80x9cTfxe2x80x9d) receptor (xe2x80x9cTf-Rxe2x80x9d). A Tf-targeted immunotoxin known as Tf-CRM107 (Johnson, V. G. et al., J. Biol Chem., 263:1295-1300 (1988)), a conjugate of transferrin (xe2x80x9cTfxe2x80x9d) and a mutant diphtheria toxin (xe2x80x9cDTxe2x80x9d) lacking receptor-binding function (Greenfield, L. et al., Science, 238:536-539 (1987), can target and kill cells expressing Tf-R, such as tumor cells The potential of Tf-CRM107 for brain tumor therapy has been explored in vitro (Johnson, V. G. et al., J. Biol Chem., 263:1295-1300 (1988), in animal models (Laske, D. W. et al., J. Neurosurg., 80:520-526 (1994)), and in patients with malignant gliomas (Laske, D. W. et al., Nat. Med., 3:1362-1368 (1997) (hereafter, xe2x80x9cLaske 1997xe2x80x9d). When delivered by high-flow (4-10 xcexcl/min) interstitial microinfusion convection-enhanced delivery (xe2x80x9cCEDxe2x80x9d) (Bobo, R. H. et al., Proc. Natl. Acad. Sci. USA, 91:2076-2080 (1994)), intratumoral infusion of Tf-CRM107 in patients with malignant brain tumors produces tumor responses (Laske 1997). When CED is used, Tf-CRM107 (140 kDa) is distributed preferentially into the interstitial space of the tumor and the surrounding brain infiltrated by tumor and circumvents the blood-brain barrier (xe2x80x9cBBBxe2x80x9d).
One factor limiting the success of Tf-CRM107 therapy is the fact that capillary endothelial cells in the brain express low levels of Tf-R (Jeffries, W. A. et al., Nature, 312:162-163 (1984)). A portion of patients receiving high doses of Tf-CRM107 display neurological deficits consistent with endothelial damage. MRI in these patients has revealed changes in the brain consistent with microvascular occlusion and/or petechial hemorrhage (Laske 1997). This vascular damage therefore limits the doses at which Tf-CRM107, and other immunoconjugates using Tf as a targeting agent, can be administered.
Lysosomotrophic amines, such as chloroquine, are used clinically to treat malaria and certain collagen diseases. These drugs accumulate in lysosomes and increase and neutralize vesicular pH (Kim, K., and Groman, N. B., J. Bacteriol., 90:1552-1556 (1965)). DT enters the cell to inhibit protein synthesis, using the low pH of endosomes and lysosomes to trigger transport into the cytosol (Sandvig, K., and Olsnes, J. Biol. Chem., 256:9068-9076 (1981); Donovan, J. J. et al., Proc. Natl. Acad. Sci. USA, 78:172-176 (1981); Sandvig, K., and Olsnes, S., J. Cell Biol., 87:828-832 (1980); Draper, R. K., and Simon, M. I., J. Cell Biol., 87:849-854 (1980). Thus, in vitro, chloroquine blocks the cytotoxicity of DT (Leppla, S. et al., J. Biol. Chem., 255:2247-2250 (1980)), and presumably that of Tf-CRM107, whose toxicity arises from the same mechanism.
Unfortunately, the very benefit that chloroquine confersxe2x80x94blocking of the toxicity of Tf-CRM107xe2x80x94also renders its use problematic in connection with Tf-CRM107 cancer therapy. The clinical utility of Tf-CRM107 is its toxicity to the target tumor cells. Therefore, if chloroquine or a like agent is co-administered with Tf-CRM107 or other immunotoxins for which lowered endosomal pH is part of the process which activates their cytotoxic function, it can be expected that the chloroquine or other agent will render the tumor cells resistant to the toxic effects of the immunotoxin, thereby reducing the desired therapeutic effect.
The invention provides methods of decreasing toxicity to a vascular endothelial cell of immunotoxins used to treat brain tumors and residual cancer cells following surgical removal of a brain tumor. The immunotoxin is administered to the brain, and contacts the ablumenal surface of the vascular endothelial cell. The method comprises contacting the lumenal surface, but not the ablumenal surface, with an endosome pH-raising agent in an amount sufficient to protect the cell from toxic effects of the immunotoxin. The endosome pH-raising agent is selected from the group consisting of a lysosomotrophic amine, a proton ionophore, and a vacuolar H+ ATPase inhibitor. The lysosomotrophic amine can be, for example, chloroquine, hydrochloroquine, mefloquine, or a congener of chloroquine. The proton ionophore can be monensin. The vacuolar H+ ATPase inhibitor can be bafilomycin A. The contacting can occur in vivo.
The immunotoxin used in the method can comprise a toxic moiety selected from the group consisting of a mutated Pseudomonas exotoxin (PE) and a mutated diphtheria toxin (DT). Where a mutated PE is chosen as the toxic moiety, preferred forms of the PE are PE4E, PE35, PE38, and PE40. Where a mutated DT is chosen as the toxic moiety, it can be a diphtheria toxin with a deletion of all or some of the native receptor-binding domain, which results in reduced non-specific binding or toxicity compared to wild-type DT, a diphtheria toxin with a substitution of an amino acid for serine at position 508 which results in reduced non-specific binding or toxicity compared to wild-type DT, a diphtheria toxin with a substitution of an amino acid for serine at position 525 which results in reduced non-specific binding or toxicity compared to wild-type DT, and a diphtheria toxin with a substitution of an amino acid other than serine for serine at position 508 and of an amino acid other than serine for serine at position 525, which substitutions result in reduced non-specific binding or toxicity compared to wild-type DT. In some preferred forms, the DT deletion mutant of the native receptor-binding domain can commence at an amino acid residue selected from residue 388 and residue 389. In a particularly preferred embodiment, the mutated DT is one in which the serine at position 525 is replaced with a phenylalanine (the DT so mutated is known as xe2x80x9cCRM107xe2x80x9d). A preferred immunotoxin is Tf-CRM107.
In preferred embodiments, the vascular endothelial cell is in the brain of a mammal. In particularly preferred embodiments, the mammal is a human. In preferred embodiments, the immunotoxin is administered directly into the brain and the endosome pH-raising agent is administered systemically. In the most preferred method, the immunotoxin is Tf-CRM107 and the endosome pH-raising agent is chloroquine.
In another set of embodiments, the invention provides methods of decreasing toxicity of an immunotoxin administered into a brain to a vascular endothelial cell in the brain, the methods comprising systemically administering an endosome pH-raising agent in an amount sufficient to decrease toxicity of the immunotoxin to the vascular endothelial cell. The endosome pH-raising agent can be a lysosomotrophic amine, a proton ionophore, or a vacuolar H+ ATPase inhibitor. The lysosomotrophic amine can be chloroquine, hydrochloroquine, mefloquine, or a congener of chloroquine. The proton ionophore is monensin. The vacuolar H+ ATPase inhibitor is bafilomycin A.
The toxic moiety can be a mutated Pseudomonas exotoxin (PE) or a mutated diphtheria toxin (DT). Where a PE based immunotoxin is used, the toxic moiety can be PE4E, PE35, PE38, and PE40. Where a DT-based immunotoxin is employed, the toxic moiety can be selected from a diphtheria toxin with a deletion of all or some of the native receptor-binding domain which results in reduced non-specific binding or toxicity compared to wild-type DT, a diphtheria toxin with a substitution of an amino acid for serine at position 508 which results in reduced non-specific binding or toxicity compared to wild-type DT, a diphtheria toxin with a substitution of an amino acid for serine at position 525 which results in reduced non-specific binding or toxicity compared to wild-type DT, and a diphtheria toxin with a substitution of an amino acid other than serine for serine at position 508 and of an amino acid other than serine for serine at position 525, which substitutions result in reduced non-specific binding or toxicity compared to wild-type DT. Further, the deletion of the native DT receptor-binding domain can commence at an amino acid residue selected from residue 388 and residue 389. In a particularly preferred embodiment, the mutated DT is one in which the serine at position 525 is replaced with a phenylalanine (the DT so mutated is known as xe2x80x9cCRM107xe2x80x9d). A preferred immunotoxin is Tf-CRM107.